Antifungal and phytotoxic activity of benzoic acid derivatives from inflorescences of Piper cumanense.

The Piperaceae family is recognized for its diverse biological properties such as antifungal, antibacterial, cytotoxic and insecticidal activities. Phytochemical investigation of inflorescences from P. cumanense led to the isolation and identification of a new 4-chromanone called oxocumanensic acid, along with five known benzoic acid derivatives. The antifungal activity was evaluated against six phytopathogenic fungi by a mycelium growth inhibition test. All compounds were active against the phytopathogenic fungi, showing a greater activity against fungi of Fusarium genus. Finally, phytotoxicity was evaluated in the extract and its chemical constituents, finding that the natural substances are less toxic than the positive control.

Effect of Different Durations of Solid-Phase Fermentation for Fireweed (Chamerion angustifolium (L.) Holub) Leaves on the Content of Polyphenols and Antioxidant Activity In Vitro.

Fireweed has recently been recognized as a plant with high antioxidant potential and phenolic content. Its leaves can be fermented to prepare an infusion with ideal antioxidant activity. The aim of this study was to investigate and to determine the influence of solid-phase fermentation of different durations on the variation of polyphenols in the leaves of fireweed. Laboratory experiments were conducted in 2017-2018. The leaves of fireweed, naturally growing, were fermented for different periods of time: not fermented (control) and fermented for 24 and 48 h. The evaluation of polyphenols and antioxidant activity in leaves was performed using high- performance liquid chromatography (HPLC). Additionally, principal component analysis was used to characterize differences in bioactive compounds between fireweed samples fermented at different durations. Solid-phase fermented leaves were characterized by higher contents of oenothein B, quercetin and benzoic acid but had lower contents of quercetin-3-O-rutinoside, luteolin and chlorogenic and gallic acids. Antioxidant activity in short- (24 h) and long-term (48 h) fermentation (compared to control) gave the highest level of regression in 2017, but in 2018 the effect was observed only with short-term fermentation and control. In conclusion, solid-phase fermentation can be used to modulate biologically active compounds in fireweed leaves.

Structural optimization and neurotrophic activity evaluation of neurotrophic gentiside derivatives.

C14 alkyl benzoate ABG001, derived from naturally occurring gentisides, was reported to exhibit neurotrophic activity which is similar to NGF (Nerve Growth Factor). In this research, ABG001 was modified by the strategy of isosteric replacement and conformational restriction with the purpose of improving the bioactivity. The cellular neurotrophic activity of those ABG001 derivatives were evaluated, among which 3-hydroxyquinolin-2-(1H)-one A3 and 4-decylphenol ester B7 displayed much better neurotrophic activity compared with ABG001, which highlights the potential of those novel scaffolds for future neurotrophic agent development.

Separation of six compounds from pigeon pea leaves by elution-extrusion counter-current chromatography.

A valid and reliable method was established to separate six compounds from pigeon pea leaves via elution-extrusion counter-current chromatography. A solvent system composed of n-hexane/methanol/formic acid aqueous solution with pH = 3 (10:6:4, v/v) was screened to achieve satisfactory isolation from the ethanol extract of pigeon pea leaves. Four compounds, 9.2 mg of compound 1 (96.8%), 3.2 mg of 2 (88.0%), 6.2 mg of 4 (94.2%) and 25.2 mg of 5 (94.2%), were obtained by conventional elution from 100 mg of the precipitation fraction, respectively. Two compounds, 14.4 mg of 3 (96.3%) and 28.1 mg of 6 (96.6%), with high K values were obtained by the subsequent extrusion procedure. The compounds 1-6 were identified as 3-methoxy-5-(2-phenylethenyl)-phenol, pinostrobin chalcone, pinostrobin, 2-hydroxy-4-methoxy-6-(2-phenylvinyl)-benzoic acid, longistylin C and cajaninstilbene acid by quadrupole time-of-flight mass spectrometry, and 1 H and 13 C NMR spectroscopy. The in vitro antiproliferation activities of compounds 1, 5 and 6 against human hepatoma cell were evaluated and the half-maximum inhibitory concentrations were acquired.

A simple and highly-available microextraction of benzoic and sorbic acids in beverages and soy sauce samples for high performance liquid chromatography with ultraviolet detection.

A simple and highly-available air-assisted dispersive liquid-liquid microextraction procedure with organic phase solidification (AA-DLLME-OPS) was developed as new approach for pretreatment of complex sample matrix. Menthol was investigated as novel extractant for the AA-DLLME-OPS. In this procedure, a cloudy solution of fine molten menthol droplets is formed when a mixture of molten menthol and aqueous sample is rapidly aspirated into a dispenser and injected into an extraction vial. In the AA-DLLME-OPS, air bubbles promote molten menthol dispersion into aqueous sample phase. Finally, the obtained mixture is cooled and menthol phase is solidified because of the low melting point, which facilitates collection for analysis. To demonstrate the efficiency of the suggested approach, the AA-DLLME-OPS procedure was applied for the HPLC-UV determination of benzoic and sorbic acids as proof-of-concept analytes in beverages and soy sauce samples. The procedure developed provides microextraction of benzoic and sorbic acids from samples with recovery from 93 to 105% and from 96 to 101%, respectively. Under optimal experimental conditions the linear detection ranges were found to be 0.1-150 mg L-1 for benzoic acid and 0.05-100 mg L-1 for sorbic acid with LODs calculated from a blank test, based on 3σ, 0.03 mg L-1 and 0.02 mg L-1, respectively.

HPTLC Autography Based Screening and Isolation of Mushroom Tyrosinase Inhibitors of European Plant Species.

In the course of this project, 133 plants were evaluated on their ability to inhibit tyrosinase, a key enzyme in melanogenesis. The screening was performed by means of a HPTLC autographic assay, resulting in the selection of three plants, Asplenium trichomanes, Pinus uncinata, and Scutellaria altissima, with promising tyrosinase inhibiting activities. With the aid of the HPTLC assay, it was not only possible to select the most interesting plant extracts, but also to monitor the activity-guided fractionation which, in a relatively short time period, led to the isolation of active principles. Benzoic acid, roseoside, and dihydrovomifoliol-O-ß-d-glucopyranoside could be identified as tyrosinase inhibitors present in P. uncinata. Globularin turned out to be the active principle of S. altissima, and 4-ethenylphenyl 6-O-(6-deoxy-α-l-mannopyranosyl)-ß-d-glucopyranoside was detected as tyrosinase inhibitor of A. trichomanes. The pure compounds were tested also in a 96 well-plate assay in order to determine their IC50 values. The lowest IC50 value (42 µm) could be obtained for globularin, whereas the other compounds, e. g., benzoic acid exhibited a rather high IC50 value (IC50 =552 µm). This stood in clear contrast to the autographic assay, but is has to be taken into account that the outcome of the autography assay is not only depending on the IC50 value of a compound, but also on the content of the respective constituent in the extract.

Separation of Benzoic and Unconjugated Acidic Components of Leonardite Humic Material Using Sequential Solid-Phase Extraction at Different pH Values as Revealed by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Correlation Nuclear Magnetic Resonance Spectroscopy.

Here, we report on sequential solid-phase extraction of leonardite hymatomelanic acid (CHM) on a non-ionic sorbent at four steadily lowered pH values: 7, 5, 3, and 2, yielding fractions with different acidic properties. Using nuclear magnetic resonance (NMR) spectroscopy and ultrahigh-resolution mass spectrometry, we revealed a gradual shift of dominating scaffolds in the fractions of CHM from reduced saturated to oxidized aromatic compounds. An increase on the average aromaticity of the CHM fractions was accompanied by a red shift in fluorescence spectra. These results were supported by heteronuclear single quantum coherence and heteronuclear multiple bond correlation NMR experiments. We have demonstrated that the CHM fraction isolated at pH 5 was dominated by aliphatic carboxyl carriers, while the pH 3 fraction was dominated by aromatic carboxyl acids. The developed fractionation technique will enable deeper insight on structure-property relationships and the design of the humic-based materials with tailored reactive properties.

Profile of Polyphenolic and Essential Oil Composition of Polish Propolis, Black Poplar and Aspens Buds.

In this work, we studied similarities and differences between 70% ethanol in water extract (70EE) and essential oils (EOs) obtained from propolis, black poplars (Populus nigra L.) and aspens (P. tremula L.) to ascertain which of these is a better indicator of the plant species used by bees to collect propolis precursors. Composition of 70EE was analyzed by UPLC-PDA-MS, while GC-MS was used to research the EOs. Principal component analyses (PCA) and calculations of Spearman's coefficient rank were used for statistical analysis. Statistical analysis exhibited correlation between chemical compositions of propolis and Populus buds' 70EE. In the case of EOs, results were less clear. Compositions of black poplars, aspens EOs and propolises have shown more variability than 70EE. Different factors such as higher instability of EOs compared to 70EE, different degradation pattern of benzyl esters to benzoic acid, differences in plant metabolism and bees' preferences may be responsible for these phenomena. Our research has therefore shown that 70EE of propolis reflected the composition of P. nigra or complex aspen⁻black poplar origin.

A new antifungal benzoic acid ester from Uvaria narum.

Uvaria narum has been used for gastrointestinal problems, jaundice, fever and skin diseases in traditional and ethnomedical practices. Our preliminary antifungal screening of various leaf extracts of U. narum revealed very good antifungal activity for its acetone extract. Active principle of U. narum leaf acetone extract was isolated by bioactivity-guided fractionation and characterised as a new molecule, 2-E-(2″-oxo-5″-acetoxy cyclopent-3″-en-1″-ylidene) ethyl benzoate, by NMR, IR and mass spectroscopic analyses. This active isolate showed very good activity against the fungal pathogen, Colletotrichum gloeosporioides.

Thin-film microextraction coupled to surface enhanced Raman scattering for the rapid detection of benzoic acid in carbonated beverages.

Benzoic acid (BA) is widely used as an antimicrobial preservative in carbonated beverages. In this study, silica gel thin-film microextraction (TFME) coupled to surface enhanced Raman scattering (SERS) was applied for the rapid detection of BA in carbonated beverages. The TFME process was performed by placing small pieces of silica gel substrate in a simple homemade device, and then the content of BA was detected using SERS after colloidal gold was uniformly dropped onto the substrate. The obtained SERS signals of BA were strong and of high reproducibility. A good linearity between the concentration of BA and the SERS signal intensity at 994cm-1 in the range of 25-500µgmL-1 was obtained under optimal experimental conditions. The detection limit value was found to be 3.6µgmL-1 and the recovery of the spiked BA was in the range 85.0-103.0%. The developed method was successfully applied to detect BA in carbonated beverage samples as the results were consistent with those using high performance liquid chromatography, suggesting that it is a rapid, convenient and sensitive way to achieve BA detection in carbonated beverages.

Isolation and characterization of metabolites from Bacillus licheniformis MH48 with antifungal activity against plant pathogens.

In this study, we isolated Bacillus licheniformis MH48 from rhizosphere soil and demonstrated that this strain shows significant antifungal activity against Rhizoctonia solani, Colletotrichum gloeosporioides, and Phytophthora capsici. Our results showed that a 50% concentration of bacterial cell-free culture filtrate of B. licheniformis MH48 shows strong activity against fungal pathogens. Benzoic acid produced by B. licheniformis MH48 was purified by various chromatographic techniques and identified by nuclear magnetic resonance and gas chromatography-mass spectrometry analysis. Benzoic acid displayed antifungal activity against R. solani and C. gloeosporides with minimum inhibitory concentration of 128 µg/mL against mycelial growth. Microscopic examination revealed that benzoic acid (50 µg/mL and 100 µg/mL) transformed C. gloeosporioides conidial morphology and inhibited conidial germination. In addition, benzoic acid (100 µg/mL and 200 µg/mL) degraded R. solani mycelia. Therefore, our results demonstrate that B. licheniformis MH48 strain shows potential for utility as a biological agent for the control of various fungal pathogens of plants.

Metabolism and antioxidant effect of malaxinic acid and its corresponding aglycone in rat blood plasma.

Malaxinic acid (MA) is a phenolic acid compound, found mainly in pear fruits (Pyrus pyrifolia N.), that is isoprenylated on the C-3 position of benzoic acid. Recently, the effects of prenylated phenolics on health have received much interest owing to their reported potent beneficial biological effects. We conducted a comparative study in rats to determine the metabolism, pharmacokinetics, and antioxidative activities of MA and its corresponding aglycone (MAA). MA and MAA were orally administered to rats (Sprague-Dawley, male, 6 weeks old) and their metabolites in plasma were analyzed. In addition, the MA metabolites in plasma were separated and the structures were confirmed via NMR and HR-MS analyses. The antioxidative activities of MA and MAA were evaluated by measuring their inhibitory effects on the 2,2'-azobis(2-amidinopropane)dihydrochloride- or copper ion-induced lipid peroxidation of rat plasma. MA was not absorbed in the intact form (the glucoside); both MA and MAA were absorbed as MAA and its metabolite form (glucuronide or sulfate). Moreover, the observed metabolite was the glucuronate of MAA rather than the glucuronide or sulfate. Concentrations of the free form of aglycone (MA administration, 4.6 ± 2.2µM; MAA administration, 7.2 ± 2.3µM) and total MAA (MA administration, 19.6 ± 4.4µM; MAA administration, 21.7 ± 3.3µM) in plasma reached a maximum at 15min after the oral administration of MA and MAA, respectively. The relative inhibitory effects on the formation of cholesteryl ester hydroperoxides in plasma collected at 15min after the oral administration of MA, MAA, and p-hydroxybenzoic acid (p-HBA) were as follows: MAA > MA ≥ p-HBA > control. Although the majority of MA and MAA is metabolized to conjugates, the compounds may contribute to the antioxidant defenses in the blood circulation owing to the presence of a phenolic hydroxyl group in the free form.

Prenylated benzoic acid derivatives from the stem of Euodia lepta.

Two new prenylated benzoic acid derivatives, leptoic acid A and (+)-S-anodendroic acid (1-2), along with one known compound, 2,2-dimethyl-2H-1-benzopyran-6-carboxylic acid (3) were isolated from the stem of Euodia lepta (spreng.) Merr. Their structures were elucidated on the basis of the chemical and spectroscopic evidence.

Ultrasound-Assisted Surfactant-Enhanced Emulsification Micro-Extraction Followed by HPLC for Determination of Preservatives in Water, Beverages and Personal Care Products.

An ultrasound-assisted surfactant-enhanced emulsification micro-extraction (UASEME) procedure has been developed for pre-concentration of benzoic acid (BA) and paraben preservatives, including methylparaben, ethylparaben, propylparaben and butylparaben, prior to high-performance liquid chromatography-ultraviolet (HPLC-UV) analysis. Separations were performed on a Lichrospher RP-18 endcapped 5 µm, using an isocratic mobile phase of 40% acetonitile, at a flow rate of 1 mL min-1 The selected UASEME conditions comprised the use of 10 mL sample extract, 125 µL 1-octanol as extraction solvent and 0.05 mmol L-1 Tween 20 as emulsifier, 0.5% sodium chloride, ultrasonication time of 6 min and centrifugation time of 10 min. Method performance demonstrated wide linear range between 0.5 and 7,000 µg L-1 (R2 > 0.9903) and limits of detection between 0.03 and 10 µg L-1, which providing the enrichment factors of 15-184. The method precision (relative standard deviation) was <7%. The developed UASEME coupled with HPLC-UV has been successfully applied to determine four paraben preservatives in various sample matrices such as water, beverages and personal care products. The recoveries in the range of 70-138.1% were obtained. However, BA could not be determined in real sample extracts.

HSCCC Separation of the Two Iridoid Glycosides and Three Phenolic Compounds from Veronica ciliata and Their in Vitro Antioxidant and Anti-Hepatocarcinoma Activities.

Five main compounds, including two iridoid glycosides (catalposide, verproside) and three phenolic compounds (luteolin, 4-hydroxy benzoic acid, 3,4-dihydroxy benzoic acid), were separated and prepared from the crude extract of Veronica ciliata by high-speed countercurrent chromatography. n-Hexane/n-butanol/water (1.5:5:5, v/v/v) was used for the separation of catalposide and verproside. n-Hexane/n-butanol/water (3:2:5, v/v/v) was used for the separation of luteolin, 4-hydroxy benzoic acid and 3,4-dihydroxy benzoic acid. The head-to-tail elution mode was used with a flow rate of 5.0 mL/min and a rotary speed of 800 rpm. Finally, a total of 1.28 mg luteolin, 6 mg 4-hydroxy benzoic acid, 2 mg 3,4-dihydroxy benzoic acid, 2 mg verproside and 10 mg catalposide with purities of 98%, 99.1%, 99.5%, 99.8% and 99%, respectively, were obtained from 200 mg of crude extract. In addition, their structure was identified using MS, ¹H-NMR and (13)C-NMR. To the best of our knowledge, this is the first report of the separation and purification of iridoid glycosides and phenolic compounds from V. ciliata by high-speed countercurrent chromatography (HSCCC). Among these compounds, luteolin, 4-hydroxy benzoic acid and 3,4-dihydroxy benzoic acid were separated from V. ciliata Fisch. for the first time. The results of the antioxidant activity show that protocatechuic acid and luteolin have strong antioxidant activity compared to 2,6-di-tert-butyl-4-methylphenol (BHT) and vitamin C (Vc). Five compounds also exhibited strong anti-hepatocarcinoma activities.

Rapid fabrication of ionic liquid-functionalized monolithic column via in-situ urea-formaldehyde polycondensation for pressurized capillary electrochromatography.

A novel strategy for rapidly fabricating ionic liquid (IL)-bonded multifunctional monolithic stationary phase has been developed by an in-situ polycondensation of urea-formaldehyde (UF) and a lab-made acylamino-functionalized IL (1-acetylamino-propyl-3-methylimidazolium bromide, [AAPMIm]Br). Two polycondensation processes of UF with 1-amino-propyl-3-methylimidazolium bromide or [AAPMIm]Br were evaluated. Several parameters including mass ratio of urea-formaldehyde, amount of [AAPMIm]Br, polycondensation time and reaction temperature were optimized, and the [AAPMIm]Br-bonded monolithic stationary phase could be rapidly synthesized in 10min with a satisfactory permeability and mechanical stability. Used for pressurized capillary electrochromatography (pCEC), a typical hydrophilic interaction (HI) retention could be obtained in the resultant [AAPMIm]Br-bonded monolith when the content of acetonitrile (ACN) in mobile phase exceeded 20%. Multiple retention mechanisms such as hydrophilic interaction (HI), hydrogen bond (HH), anion-exchange and cation-exclude interactions, were acheived in the [AAPMIm]Br-bonded monolith. Various polar compounds including phenols, benzoic acid and its homologues, and enkephalins have been well separated and thus demonstrated a satisfactory separation performance of the obtained monolith. A facile access is lighted for rapid preparation of ionic liquid-bonded monoliths with multiple retention mechanisms for pCEC.

Effects of pH mismatch between the two dimensions of reversed-phase×reversed-phase two-dimensional separations on second dimension separation quality for ionogenic compounds-I. Carboxylic acids.

Two persistent impediments to wider adoption of two-dimensional liquid chromatography (2D-LC) are the perceptions that 2D methods are generally less sensitive than 1D ones, and that coupling of certain separation modes in a 2D system is difficult because of the negative impact of the effluent of the first separation on the second separation. In this work we address these problems in the specific case where reversed-phase separations are used in both dimensions of a 2D-LC system, but the pH is varied such that the ionization state of carboxylic acid analytes is different (i.e., neutral or negatively charged, in eluents buffered at pH 2 or 7) in the two columns. We first demonstrate that the effect of first dimension ((1)D) effluent on the performance of second dimension ((2)D) separation of ionogenic solutes is much more serious than it is for neutral compounds where the pH of the eluent does not play a role in retention. We have systematically varied the properties of the sample solution injected into the (2)D column (i.e., the (1)D effluent), as well as the (2)D eluent, with the goal of establishing guidelines for conditions that yield acceptable (2)D performance. We find that the organic solvent content of the (1)D effluent and (2)D eluent is not as important as the buffer concentrations in these two solutions, and that the greater the ratio of buffer concentration in the (1)D effluent relative to the (2)D eluent, the smaller the volume one can inject into the (2)D column before dramatic peak splitting occurs. We have then used the information from these simple experiments to guide both 1D experiments that mimic the (2)D separation, and actual 2D separations, to demonstrate that online adjustment of the properties of the (1)D effluent by dilution with a buffered solvent prior to injection into the (2)D column is a very effective solution to the pH mismatch problem. We find that when the buffer capacity of the diluent is high enough to effectively titrate the (1)D effluent such that its pH approaches that of the (2)D eluent, excellent (2)D peak shape is obtained for the carboxylic acid analytes, even when the volume of injected sample solution exceeds the (2)D column volume.

Polar compounds from Parentucellia viscosa (L.) Caruel from Sardinia.

In this work, we examined a sample of Parentucellia viscosa from Sardinia island, showing similarities and differences of the secondary metabolite content with respect to previous study conducted on an accession of continental origin, and also with Bellardia trixago, a species very close to Parentucellia. In this aspect, the proximity between these species, previously stated by a phylogenetic study, was confirmed by a phytochemical approach. Within the non-iridoidic fraction a chiral polyol (mannitol) and two aromatic acids (benzoic acid and gallic acid) were evidenced. These two acids are not common in Scropulariaceae since they have been reported only from a few species, and it is worth to note that gallic acid was never reported from the Orobanchaceae family.

A novel ionic liquid-modified organic-polymer monolith as the sorbent for in-tube solid-phase microextraction of acidic food additives.

A novel ionic liquid-modified organic-polymer monolithic capillary column was prepared and used for in-tube solid-phase microextraction (SPME) of acidic food additives. The primary amino group of 1-aminopropyl-3-methylimidazolium chloride was reacted with the epoxide group of glycidyl methacrylate. The as-prepared new monomer was then copolymerized in situ with acrylamide and N,N'-methylenebisacrylamide in the presence of polyethylene glycol (PEG)-8000 and PEG-10,000 as porogens. The extraction performance of the developed monolithic sorbent was evaluated for benzoic acid, 3-hydroxybenzoic acid, cinnamic acid, 2,4-dichlorophenoxyacetic acid, and 3-(trifluoromethyl)-cinnamic acid. Such a sorbent, bearing hydrophobic and anion-exchange groups, had high extraction efficiency towards the test compounds. The adsorption capacities for the analytes dissolved in water ranged from 0.18 to 1.74 µg cm(-1). Good linear calibration curves (R(2) > 0.99) were obtained, and the limits of detection (S/N = 3) for the analytes were found to be in the range 1.2-13.5 ng mL(-1). The recoveries of five acidic food additives spiked in Coca-Cola beverage samples ranged from 85.4 % to 98.3 %, with RSD less than 6.9 %. The excellent applicability of the ionic liquid (IL)-modified monolithic column was further tested by the determination of benzoic acid content in Sprite samples, further illustrating its good potential for analyzing food additives in complex samples.

Antibacterial prenylbenzoic acid derivatives from Anodendron formicinum.

A phytochemical investigation on the stems of Anodendron formicinum led to the isolation of eight prenylbenzoic acid derivatives. Three of these were new compounds, designated as formicinuosides A (1), B (2), and C (3). Their structures were elucidated on the basis of extensive spectroscopic analysis, as well as by comparison with the reported spectroscopic data. This is the first report of chemical constituents from A. formicinum and their antimicrobial activities. Among the isolated compounds, compounds 4, 6 and 8 showed significant antibacterial activities against Providensia smartii with MIC values of 0.781 µg/mL. Moreover, compound 8 showed remarkable antibacterial activity against Escherichia coli with MIC value of 0.781 µg/mL.